Anti-viral composition and kit and use for treating rotavirus infection and diarrhea

ABSTRACT

An anti-diarrheic product comprises a foodstuff and an anti-rotaviral agent such as human defatted fat globule membranes, the human milk macromolecular fraction, skim milk, curd, whey, the milk mucin complex, the 46 Kd app.MW glycoprotein, a polypeptide having an amino acid sequence having the rotavirus binding specificity of the 46 Kd app.MW HMFG glycoprotein or mixtures thereof. An anti-diarrheal kit comprises the anti-rotaviral agent of this invention and instructions for its use, and optionally a foodstuff. A method for inhibiting or countering rotavirus infection in mammalian cells comprises contacting the cells with an anti-rotaviral amount of the agent of this invention. A therapeutic method for inhibiting the onset of or countering rotavirus infection comprises administering to a subject afflicted with or at risk for rotavirus infections such as those occuring, for example, in infants and children (infantile gasteroenteritis) and immunodeficient persons or those who have received a transplant, an anti-rotaviral effective amount of the agent of this invention.

This invention was made with at least partial Government funding underGrant Nos. R01 DK 33089, CA 39932, CA 42767 HD 13021 from the NationalInstitutes of Health. The United States government may have rights inthis invention.

This application is a continuation of U.S. patent application Ser. No.08/378,865, filed Jan. 23, 1995, which issued as U.S. Pat. No.5,505,955, which is a continuation of U.S. Ser. No. 07/969,949, filedOct. 30, 1992 by the same inventors, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a prophylactic and therapeutic composition forinhibiting rotavirus infection, and more particularly to a method ofpreventing and treating diseases or conditions associated with, orrequiring, rotavirus infection, using defatted human milk globules(HMFGs), milk macromolecular fraction, curd, whey, the human milk mucincomplex or a polypeptide having the rotavirus-binding specificity of the46 Kdalton (Kd) app.MW HMFG glycoprotein. The present method finds itsapplication in the prevention and treatment of diseases such asinfantile gastroenteritis, and diarrheal conditions that afflictimmunodeficient patients, the elderly and travelers.

2. Description of the Background

Gastroenteritis and diarrhea have been linked to rotavirus infection ina variety of clinical settings. In many cases the population afflictedby these diseases are the very young, the elderly and theimmunocompromised. Acute infectious gastrointestinal diseases, forexample, are a major cause of illness and death in infants and youngchildren throughout the world. In the developing countries, infectiousgastrointestinal diseases are estimated to cause up to 12,000 deaths perday. Diarrheal disease is also an important health problem in thedeveloped countries. In the U.S., over 200,000 children under 5 years ofage are hospitalized each year with acute diarrheal disease. Thisresults in nearly 880,000 in-patient hospital days, over 500 deaths, andalmost one billion dollars of in-patient costs per year.

Although various vital, bacterial, and parasitic agents are suspected ofcausing acute infectious gastroenteritis, rotaviruses have beenidentified as the most important viral agent of gastroenteritis, e.g,,in children living in both developed and developing countries.Prospective studies indicate that in the U.S. rotaviruses account foraround 2.9 million yearly episodes of diarrhea leading to 22,000 annualhospitalizations of children less than 5 years old. Rotaviruses havealso been implicated as the causative agent of diarrheal outbreaksoccurring in nursing homes, day care centers and during travel, andresulting from adult contacts with sick children. Additionally,rotaviruses have been linked with the occurrence of diarrhea in patientsundergoing bone marrow transplants and suffering from variousimmunodeficient conditions.

Throughout history, breast-feeding infants were shown to be somewhatprotected against enteric infection by pathogens in general whencompared with bottle fed infants. In addition, breast-feeding was alsoshown to lower the incidence of enteric diseases, such as necrotizingenterocolitis of infancy, which may be infectious in origin but whichhave not been associated with a single etiologic agent. More recently,studies of children living in developing as well as developed countriessuch as Great Britain and the U.S. have shown that breast-fed infantsundergo substantially fewer episodes of gastroenteritis than bottle-fedinfants. In some studies breast-feeding was shown to lessen the severityof diarrhea and vomiting associated with rotavirus infection inhospitalized children, but not to provide total protection againstinfection in general, and serious episodes of rotavirus infection inparticular. However, no single factor was found to be responsible forthis effect in spite of the fact that antibodies were suspected of beinginvolved in the effect. The level of anti-rotavirus antibody in humanmilk was found not to correlate with the degree of protection affordedby the milk. This suggested that non-immunoglobulin factors may play arole in the protective process. Among the non-immunoglobulin factorsthat have been implicated in this phenomenon are lipids, α-interferonand trypsin inhibitors, among others. It has also been suggested thatsome of these substances are possible inhibitors of viral replicationand of microorganisms in general. A factor isolated from a milk fractionfree of fat and cells was shown to inhibit the infectivity ofrespiratory syncytial virus (RSV). The factor has a molecular weightgreater than 400 Kd app.MW, and is distinct from the present agents.

The growth of rotavirus and its infectivity were studied in varioussystems. Rotavirus replication was shown recently to be inhibited byavian egg and bovine submaxillary gland glycoproteins in cell culture.These glycoproteins bind to the virus and their activity requires sialicacid and proceeds by interference with the binding of the virus tocellular receptors. Intestinal brush border membranes were also shown tobind rotavirus by attachment to glycoproteins.

Human milk fat globules (HMFG) are obtained from the cream fractions ofmilk, and have been utilized to prepare polyclonal and monoclonalantibodies for use in the diagnosis of breast cancer. Both, anti-HMFGand anti-breast tumor monoclonal antibodies with specificities fordifferent epitopes of the mucin complex have been produced. Theanti-HMFG monoclonal antibodies were used to identify a large molecularweight mucin-like complex called non-penetrating glycoprotein (NPGP) onthe surface of breast epithelial

The human milk mucin is a highly glycosylated macromolecular complexconsisting of 50% carbohydrate, most of which is O-linked in addition tothe mucin molecule, this complex contains a disulfide-linked 70 Kdapparent molecular weight (app.MW) glycoprotein and a 46 Kd app.MWglycoprotein. Monoclonal antibodies raised against the 70 Kd app.MW and46 Kd app.MW glycoproteins have also been produced. The 46 Kd app.MW and70 Kd app.MW glycoproteins are found in the serum of breast cancerpatients and may thus be used as markers for breast cancer. The 70 Kdapp.MW glycoprotein, in particular, was found to co-purify with theintact mucin complex and to be linked to the mucin complex throughdisulfide bonds, making it a suspect linker protein of this complex onthe breast epithelial surface.

The structure of the polypeptide associated with the 70 Kd app.MWglycoprotein was determined by, cDNA cloning. A partial amino acidsequence of the 70 Kd app.MW polypeptide has been reported. (Larocca, D.D., et al., Cancer Research 50:5925-5930 (1990)).

The 46 Kd app.MW glycoprotein and its immune complexes were detected inthe serum of breast cancer patients using monoclonal antibodies againstthe glycoprotein. In addition, an increase in the levels of the 46 Kdapp.MW glycoprotein in the patients' serum was also found to beassociated with the advent of tumors. The structure of the 46 Kd app.MWglycoprotein and the areinc acid sequence of its polypeptide have beendescribed. Also known are the anti-neoplastic and diagnostic use of thisglycoprotein and its polypeptide as well as the corresponding DNA andRNA sequences of the polypeptide. (Larocca, D. D., et al., CancerResearch 51:4944-4998 (1991 )), the text of which relating to thepreparation and characterization of the 46 Kd app.MW glycoprotein isincorporated herein by reference.

The recognition of the importance of rotaviral infection, and of itsepidermiological and economic consequences, has led to a substantialeffort directed at its prevention by means of active immunization.However, current vaccine regimens have displayed poor efficacy.

Accordingly, there still is a need for a patent and effective treatmentfor rotaviral infection as well as its prophylaxis, substantiallylacking detrimental side effects.

SUMMARY OF THE INVENTION

This invention relates to an anti-diarrheic product, comprising

a foodstuff; and

an anti-rotaviral infection effective amount of an agent selected fromthe group consisting of defatted human milk fat globules, the human milkmucin-70 Kd app.MW glycoprotein-46 Kd app.MW glycoprotein complex and apolypeptide comprising an amino acid sequence having therotavirus-binding specificity of the 46 Kd app.MW HMFG glycoprotein.

Also part of this invention is an anti-diarrheic kit, comprising ananti-diarrheic composition comprising an anti-rotavirus agent selectedfrom the group consisting of defatted human milk fat globules, skimmilk, the human milk macromolecular fraction, whey, the human milk mucin70-Kd app.MW glycoprotein-46 Kd app.MW gIycoprotein complex, the 46 Kdapp.MW glycoprotein, a polypeptide having the rotavirus-bindingspecificity of the 46 Kd app.MW HMFG glycoprotein, and mixtures thereof,alone or with a foodstuff and/or a pharmaceutically-acceptable carrier;and instructions for use of the kit.

This invention also relates to a method of retarding or counteringrotavirus infection of a mammalian cell comprising contacting the cellwith an anti-rotavirus infection effective amount of an agent selectedfrom the group consisting of defatted human milk fat globules, skimmilk, the human milk macromolecular fraction, whey, the human milk mucin70 Kd app.MW glycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kdapp.MW glycoprotein, a polypeptide comprising an amino acid sequencehaving the rotavirus-binding specificity of the 46 Kd app.MW HMFGglycoprotein,and mixtures thereof.

This invention relates as well to a method of retarding or counteringrotavirus infection of a subject's cells comprising administering to asubject at risk of or afflicted with a rotavirus infection ananti-rotavirus effective amount of an agent selected from the groupconsisting of defatted human milk fat globules, skim milk, the humanmilk macromolecular fraction, whey, the human milk mucin-70 Kd app.MWglycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kd app.MWglycoprotein, a polypeptide comprising an amino acid sequence having therotavirus-binding specificity of the 46 Kd app.MW HMFG glycoprotein, andmixtures thereof; and optionally, a pharmaceutically acceptable carrier,and/or a foodstuff.

Other objects, advantages and features of the present invention willbecome apparent to those skilled in the art from the followingdiscussions.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention arose from a desire by the inventors to improve onprophylactic and therapeutic methods for treating diarrheal conditionsassociated with rotavirus infection in mammals, especially in humans.More particularly, the present invention provides an effective andpotent agent for the treatment of gastroenteritis, and/or diarrhea,associated with a variety of conditions linked to rotavirus infectionsuch as infantile gastroenteritis, and some types of diarrhea prevalentin nursing home and day care centers, and afflicting travelers andadults exposed to sick children and patients subjected to bone marrowtransplant, persons with genetic immune deficiencies, those afflictedwith acquired immune deficient diseases such as AIDS, and those who'simmune systems are suppressed by drug administration and immunodeficientpatients in general. In the general population, immunodeficient patientsare particularly at risk for rotavirus infection, and where afflicted byit, are disadvantaged for mounting an immunological response to thevirus.

This invention relies on the finding by the inventors that an agentassociated with the human milk fat globule membrane, also present inskim milk, and the macromolecular fraction, has potent anti-rotaviralactivity. The agent of this invention prevents and retards rotavirusinfections and is effective in the treatment of diarrhea. Moreover, theagent of this invention is devoid of the drawbacks and side effects ofother known therapies.

The method of the invention provides significant advantages over priorart methods for the treatment of diarrhea. The prior art has focussed onimmunologic methods in the search for a therapy against diarrhea causedby infectious agents. Immunologically based methods, however, areineffective for treating immunodeficient individuals who cannot musterthe needed immunological response to fight the pressure of the virus.Preventative vaccine therapies, for example, fail to elicit an adequateimmunological response in such individuals. In addition, vaccinationagainst rotavirus infection is somewhat ineffective and impractical dueto the existence of multiple strains of rotaviruses. Another methodapplied to the treatment of diarrhea by the prior art is theadministration of anti-rotavirus serum. This is not a method of choicefor treating gastrointestinal infections because of the limitedavailability of human immune sera, and the risks associated with usinghuman products that may be a source of contamination with viruses suchas the HIV and hepatitis viruses. Moreover, the substitution of animalsera for human sera would most likely elicit an immunological hostrejection. Also, the utilization of a single serum will fail to providecomplete protection against the multiple strains of rotaviruses.Furthermore, since large quantities of anti-rotaviral immune serum areneeded for administration by mouth, the cost of producing these largequantities, thus, makes its mass production impractical.

The administration of the agent of this invention provides a highlyeffective anti-diarrheal effect without the drawbacks produced by otheranti-viral agents such as α-interferon, ribonucleases, milk lipids andtrypsin inhibitors.

One advantage afforded by the non-immunological milk agent of theinvention over prior art products relies on the fact that the presentagents bind to many strains of human rotaviruses as well as torotaviruses of other species, e.g., mouse and simian, inhibiting viralreplication of all strains tested, and prevent rotavirus-associateddiarrhea in vivo. Thus, the agent of this invention provides protectionfor the treatment of, e.g., diarrhea caused by a wide range of rotavirusstrains.

The components of human milk found to be effective in the treatment ofgastroenteritis and diarrhea provided herein are different from theprior art agents discussed above. For example, the human fat globulemembrane, the mucin complex and particularly the 46 Kd app.MW HMFGglycoprotein are readily distinguishable from other antiviral componentsof milk such as lipids, immunoglobulins and oligosaccharides. Thedefatted human milk fat globule membrane or the dHMFG macromolecularfraction, the whole milk mucin complex and the 46 Kd app.MW polypeptidewere shown by the present inventors to physically bind to rotaviruses inthe absence of immunoglobulins, lipids and oligosaccharides. The 46 Kdapp.MW HFMG glycoprotein and the milk mucin complex are different fromother polypeptides that are present in milk such as immunoglobulins,α-interferon, and the like.

The milk mucin complex is found in the acidic fractions of milk obtainedby isoelectric focussing, and is easily separated by this method fromimmunoglobulins which are present in the basic fractions of milk. Themilk mucin complex is, thus, also different from other antiviral agentspresent in milk.

Upon fractionation of the human milk fat globules, human milk globulemembrane which is the globule's macromolecular component, and its acidicprotein fraction retain The anti-rotaviral activity. When the defattedmilk fat globule fraction is separated into different fractions, theanti-rotaviral activity of human milk remains mostly with the mucincomplex. However, when the mucin complex is separated into itscomponents the highest anti-rotavirus activity is found with 46 Kdapp.MW glycoprotein. The 46 Kd app.MW glycoprotein preferentially bindssimian and human rotaviruses when compared to the 70 Kd app.MWglycoprotein and the milk mucin depleted of the 46 Kd app.MWglycoprotein.

The human milk fat globules, the macromolecular fraction and the milkmucin complex which among other fractions contain the 46 Kd app.MWglycoprotein, and the 46 Kd app.MW glycoprotein, were all found by thepresent inventors to inhibit the infection by rotavirus of human andsimian origin of cultured mammalian cells.

The mucin complex was shown by the present inventors to inhibitrotavirus infectivity with a 3000 fold greater specific activity thanwhole milk. These results are unexpected based on the ambiguous reportsrelating to the effect of human milk on rotavirus, and the reportedinhibitory effects of other milk components on this virus.

The human 46 Kd app.MW glycoprotein was also shown by the inventors tobind to cells and cell extracts that are infected with a humanrotavirus. Human strains of the virus, such as RRV, Wa, DS-1, P andST-3, bind to the 46 Kd app.MW glycoprotein in essentially equivalentamounts thereto. Moreover, the inventors found that when sialic acid wasremoved from the 46 Kd app.MW glycoprotein, its binding to vitalinfected cells was substantially reduced. This reduction in binding ofthe 46 Kd app.MW polypeptide to rotavirus infected cells was found to bein the range of 30 to 60%. Thus, sialic acid may be required for the 46Kd app.MW glycoprotein to retain its binding activity as well asanti-viral activity. Moreover, it is also possible that theanti-rotavirus activity of milk mucins from other sources lacking sialicacid may be enhanced by sialylation.

The inventors have also shown that the agent of this invention inhibitsthe in vitro infection of cells by rotaviruses as well asgastroenteritis induced by rotaviruses in an animal model. For instance,the administration of a murine rotavirus (EDIM) to suckling mice, causeda 100% incidence of diarrhea in the mice. However, the simultaneousadministration of the virus and the human milk macromolecular or acidicglycoprotein fraction to the suckling mice, reduced the symptoms ofdiarrhea by 90%. In contradistinction, when a bovine milk-based formulaor a control medium were administered instead, the rotavirus activityand the diarrheal symptoms remained undiminished.

The purification of the various components of the human milk fatglobules may be conducted as described in the art, or by affinitypurification as shown below. The agent of this invention is easilyprepared for clinical use. Human breast milk may be readily fractionatedby published methods into a macromolecular component comprising the fatglobule membrane. This component is distinct from oligosaccharides,lipids, immunoglobulins and other small proteins contained in milk.Likewise, whole human milk, the macromolecular fraction, and the fatglobules may be defatted to produce fat globule membranes.

The macromolecularfraction containing the milk mucin complex may beobtained by lipid extraction of fatty milk as described by Newburg, D.S., et al. (Newburg, D. S., et al, Pediatric Res. 31:22-28(1992)). Theacidic glycoprotein fraction of milk may be obtained by isoelectricfocusing as described by Yolken, R. M., et al. (Yolken, R. M., et al, J.Clin. Investigation 90:(1992)). Both these fractions have anti-rotavirusactivities that are, respectively, 3 and 38 times greater than wholemilk. The milk mucin complex may be affinity-purified in accordance withthis invention or obtained as previously described (Ceriani et al.,P.N.A.S.(USA) 74:582-589 (1977)). Natural skim milk may be prepared bycentrifuging unfrozen fresh milk, and removing the cream fraction thatcontains intact milk fat globules. When fresh milk is frozen and thawed,especially several times, sonicated, allowed to stand for a period oftime, or exposed to temperature, the fat globules are generallydisrupted. When the fat layer is then separated from the remainder or"processed skim milk", it contains mainly the lipid fraction of thecream (butter consisting of mainly triglycerides), while the milk fatglobule membranes, the 70 Kd app.MW and the 46 Kd app.MW.HMFGglycoproteins are now mainly in the "processed skim milk". However, theamount is greatly increased in the "processed skim milk", the amountincreasing with more vigorously freezing and thawing and/or sonication.Both the natural and the processed skim milk have anti-rotavirusactivity, with the latter evidencing higher activity. Curds and whey maybe prepared as is known in the art, and will contain a certainproportion of the described components that have anti-rotavirusactivity.

The milk mucin complex, in turn, may be further purified from themembranes using monoclonal antibodies as described herein, and the 46 Kdapp.MW glycoprotein may be separated from the milk mucin complex. Thesecomponents are separable by traditional chromatographic and/orelectrophoretic methods. The presence and identities of the componentsof the human milk mucin complex are readily determined using available,specific monoctonal antibodies.

The gene encoding the 46 Kd app.MW polypeptide being available, the geneproduct and variations thereof may be prepared by recombinant technologyand expressed in recombinant microorganisms as described by Larocca etal. (Larocca et al. Cancer Res. 51:4994-4998 (1991); Larocca et al.Hybridoma 11:191-201 (1992); Larroca, et.al., "Molecular Cloning andeExpression of Breast Mucin Associated Antigens"", in Breast Epit.helial Antigens, p. 36, Plenum Press, Ceriani, R. L., ed, N.Y., N.Y.(1991)). The amino acid sequence of the 46 Kd app.MW polypeptide isunrelated to any known immunoglobulin but was found to have significanthomology to human epithelial cell proteins and the C1 C2 domains of thehuman clotting factors V and VIII, a mouse milk fat globule 67 Kd app.MWprotein MFG-E8, discoidin of amoebae, and the A5 antigen of xenopusbrain, among others.

Polypeptides having the iotavirus binding characteristics of the HMFG 46Kd app.MW glycoprotein component of the mucin complex may be preparedsynthetically, by sequencing or by adding a stop codon at a desiredplace in the DNA encoding the protein, by methods known in the art, orby purification from human milk of the 46 Kd app.MW glycoprotein andsubsequent partial hydrolysis.

The synthetic polypeptide having the described characteristics may beprepared in different; lengths by alteration of the DNA sequenceencoding it and adding a stop codon where desired, as is known in theart, and expression of the thus altered gene or fragments thereof. Thegene encoding the 46 Kd app.MW polypeptide has been cloned and partiallysequenced as discussed above.

The novel anti-rotaviral agent of this invention is suitable for use inmost instances of rotavirus infection, and particularly in cases whereother therapies are either ineffective or clinically contraindicated.

The agent of this invention exhibits additional advantages for thetreatment of infants and children since, as already indicated, itscomponents are normal constituents of human milk and the human diet. Thepresent agent is thus unlikely to elicit toxic, immunological orallergic reactions in treated subjects. Because these agents areinnocuous to the human body, the invention may be used withoutintervention of skilled medical personnel, for example, by adding it tofoodstuffs, and the like, that are normally sold over-the-counter inconvenience stores or as food supplements available in grocery stores.This is a particular advantage for treating travellers or populations inunderdeveloped countries where medical services are in short supply.

The agent of this invention may be administered in combination withother treatments, such as immune therapy, particularly treatments thatact by independent mechanisms, to thereby provide a multi-pronged attackon the virus. Other anti-rotaviral treatments may be combined with thepresent agent to provide a treatment compatible with other clinicalneeds of a patient, as well. For example, other milk components, such asoligosaccharides, α-interferon and trypsin inhibitors, known to haveanti-microbial and anti-viral activity, may be combined with the presentagent.

The inventors have found that components of human milk other than thoseencompassed by the invention failed to inhibit rotavirus infection incell cultures. These agents, prepared by methods described in the art,include lipids, gangliosides, polar neutral glycolipids, non-polarglycolipids, triglycerides and fatty acids and neutral, acidic and totaloligosaccharides.

The agent of the invention may be used alone, with a carrier or as anadditive to a foodstuff, or in other compositions suitable for humanconsumption.

Thus, this invention provides an anti-diarrheic product, comprising

a foodstuff; and

an anti-rotaviral effective amount of an agent selected from the groupconsisting of defatted human milk fat globules, skim milk, the humanmilk macromolecular fraction, curd, whey, the human milk mucin-70 Kdapp.MW glycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kd app.MWHMFG glycoprotein, a polypeptide comprising an amino acid sequencehaving the rotavirus-binding characteristics of the 46 Kd app.MW HMFGglycoprotein, and mixtures thereof. Each agent may be used alone orcombined with one or more of the agents provided herein, or furthercombined with a foodstuff or food supplement for self-administration.

The agent of this invention may also be provided in a composition withother components including, but not restricted to, vitamin supplements,mineral additives, other nutritional additives, buffers, salts,flavoring compounds, diluents, thickeners, emulsifiers, preservatives,and anti-oxidants, such as would be familiar to a person skilled in theart, as would the amounts they are added in to the composition.

The anti-diarrheic composition or product may also comprise a bindersuch as gum tragacanth, acacia, corn starch or gelatin, excipients suchas dicalcium phosphate, anti-clumping agents such as corn starch, potatostarch, alginic acid and the like, lubricants such as magnesiumstearate, sweetening agents such as sucrose, lactose or saccharin,flavoring agents such as peppermint, orange, wintergreen or cherryflavoring as well as other known artificial and natural flavoringcompounds. Sustained-release preparations and formulations are alsowithin the confines of this invention, and may contain furtheringredients as is know in the art.

A coated composition, or otherwise modified forms of the preparation arealso contemplated herein such as coatings of shellac, gelatin, sugar andthe like. Any material added to this product should bepharmaceutically-acceptable and substantially non-toxic in the amountsemployed.

Other excipients may be added to the formulation such as those utilizedfor the production of ingestible tablets, troches, capsules, elixirs,suspensions, syrups and wafers, among others and the product may then beprovided in these forms.

In one preferred embodiment, the product of the invention comprises the46 Kd app.MW HMFG glycoprotein. The. glycoprotein may be compounded withother anti-viral human milk components as well as other anti-viral andanti-microbial agents as indicated above. In another preferredembodiment, the product comprises the mucin complex or mixtures thereof.

The agent of this invention may be present in the anti-diarrheic productin an amount of about 0.01 to 99.9 wt % of the composition, andpreferably about 0.1 to 20.0 wt %. However, other amounts of the agentmay also be present in the product. The amount of the agent in theanti-diarrheic product may be varied, and/or the frequency ofadministration increased, depending on the severity of the infection,the general health and nutritional status of the subject, and whether ornot other anti-rotavirus agents are being administered as well.

Foodstuffs suitable for use in the anti-diarrheic product of theinvention are milk, juices, cereals, chewing gum, crackers, candies,meats, vegetables and fruits, blended or otherwise as baby food forexample, and cookies, among others.

In another embodiment, the foodstuff of the product provided herein maybe infant formula, milk, milk substitutes, baby foods, rehydrationformula, and vitamin supplements, among others. This product may bespecifically formulated for the palate of youngsters, when applied tothe treatment of infants or small children.

This invention also provides an anti-diarrheic kit, comprising ananti-diarrheic composition comprising an agent selected from the groupconsisting of defatted human milk fat globules, skim milk, the humanmilk macromolecular fraction, curd, whey, the human milk mucin-70 Kd app.MW glycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kd app.MWglycoprotein, a polypeptide comprising an amino acid sequence having therotavirus-binding specificity of the 46 Kd app.MW HMFG glycoprotein, andmixtures thereof, and a pharmaceutically acceptable carrier; andinstructions for use of the kit.

The anti-diarrheic composition of this kit may be administered in anamount of the anti-diarrheic product of about 0.1 to 1000 mg/kg bodyweight/day, and more preferably about 1 to 50 mglkg body weight/day.Other amounts, however, may also be administered. It is understood thatthe more active fractions, such as the 46 Kd app.MW glycoprotein may beadministered at a lower dose, whereas the lesser active fractions suchas the defatted milk fat globule may be administered at a higher dose.Other amounts may also be administered. This kit is formulated for thetherapeutic treatment of subjects afflicted with or at risk of diarrhealconditions associated with rotaviral infection. The anti-diarrheiccomposition may also comprise vitamin supplements, mineral additives,other nutritional additives, salts, buffers, flavoring compounds,diluents, thickeners, emulsifiers, preservatives, and anti-oxidants,such as would be familiar to a person skilled in the art. Includedwithin the invention, is an embodiment wherein the above anti-diarrheiccompositions further comprise varying amounts of other components suchas foodstuffs. Suitable are all kinds of foods including milk and milksupplements. The anti-diarrheic composition or the product of theinvention may also be modified to include varying amounts of water andingredients suitable to the clinical needs of the subject.

The anti-diarrheic composition may be mixed with a drink (liquid) or afoodstuff for self-administration. The composition may be added in ananti-rotaviral amount, and may be provided in bulk or in unit form.

This invention also provides an anti-diarrheic kit that comprises inseparate, sterile containers

a foodstuff; and

an anti-rotaviral effective amount of an agent selected from the groupconsisting of defatted human milk fat globules, skim milk, the humanmilk macromolecular fraction, curd whey, the human milk mucin-70 Kdapp.MW glycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kd app.MWglycoprotein, a polypeptide comprising an amino acid sequence having therotavirus-binding specificity of the 46 Kd app.MW HMFG glycoprotein andmixtures thereof, and optionally a pharmaceutically-acceptable carrier;and instructions for use of the kit.

For purposes of identification of the components, the apparent molecularweight (app.MW) of the glycoproteins of the invention may be determinedby SDS-polyacrylamide gel electrophoresis using standard techniquesdescribed in the art. For example, defatted human milk fat globulemembranes may be dissolved in a solution containing 1% sodium dodecylchloride (SDC) and heated to disolve the glycoproteins, applied to a3-30% polyacrylamide gel and electrophoresed with appropriate molecularweight standards run in a parallel lane, the apparent molecular weight(app.MW) of the mucin complex obtained is approximately 400,000 Kdapp.MW or greater. The apparent molecular weights of other proteins maybe determined in a similar manner. The 46 Kd and the 70 Kd app.MWglycoproteins associated with the milk mucin complex may also beidentified by binding to the specific monoclonal antibodies Mc16 andMc13, respectively (Larocca et al., Cancer Res. 51:4994 (1991); Petersonet al., Hybridoma 9:221-235 (1990), supra). The milk mucin, alsoreferred to as breast mucin, may be identified in the complex by bindingto the monoclonal antibody Mc5 described by Peterson, J. A., et al.(Peterson, J. A., et al., Hybridoma (1990), supra). If the defattedhuman milk fat globule is disolved in SDS under reducing conditions suchas in the presence of 0.5% beta-mercaptoethanol, the 70 Kd app.MWglycoprotein runs as a doublet with an apparent molecular weight of 70.Kd, that may be further identified by binding to the monoclonalantibodies Mc13 and McR2. The 46 Kd app.MW glycoprotein under the sameconditions, appears as a doublet with an apparent molecular weight of 46Kd, as identified by binding to the monoclonal antibody Mc16 describedby Larocca, et al. (Larocca et al., Cancer Res. 51:4994 (1991), supra).The milk mucin, under reducing conditions, is seen as a band ofapproximate 400,000 Kd apparent molecular weight and may be identifiedby binding to the monoclonal antibody Mc5 described by Peterson, J. A.,et al. (Peterson, J., et al., Hybridoma (1990), supra). If the milkmucin, the 70 Kd app.MW glycoprotein, and the 46 Kd app.MW glycoproteinare treated to remove oligosaccharides, their apparent molecularweights, as determined by polyacrylamide gel electrophoresis, appear todecrease.

This invention additionally provides a method for retarding the onsetof, or countering, rotavirus infection of a mammalian cell comprisingcontacting the cell in a nutrient medium with an anti-rotaviralinfection effective amount of an agent selected from the groupconsisting of defatted human milk fat globules, skim milk, the humanmilk marcomolecullar fraction, curd, whey, the human mucin-70 Kd app.MWglycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kd app.MWglycoprotein, a polypeptide comprising an amino acid sequence having therotavirus-binding specificity of the 46 Kd app.MW HMFG glycoprotein andmixtures thereof.

In one preferred embodiment of the invention, the anti-diarrheic agentcomprises the 46 Kd app.MW HMFG glycoprotein in another embodiment, theagent comprises the mucin complex. Both of these agents may beadministered alone and/or with defatted human milk fat globules, and/orwhey, and/or curd, and/or skim milk, and/or the HMFG macromolecularcomponent, and/or the 46 Kd app.MW HMFG glycoprotein, and/or apolypeptide having the rotavirus-binding specificity of the about 46 Kdapp.MW HMFG glycoprotein and/or mixtures Thereof. Although the completeremoval of glycosides from the mucin complex was shown to reduce theanti-rotavirus activity of the glycoprotein by at least 40-60%, agentshaving varying levels of glycosylation may be used, since they retainsome activity.

This invention also provides a method of retarding the onset of, orcountering, rotavirus infection of a subject's cells comprisingadministering to a subject at risk for, or suffering from, rotavirusinfection an anti-rotavirus effective amount of the agent of thisinvention or mixtures thereof, or a composition comprising the agent ofthe invention and/or a pharmaceutically-acceptable carrier and/or afoodstuff and/or other additives as described above. The composition mayincorporate other anti-vital or anti-microbial agents, as suitable foreffective treatment of a rotavirus infection taking into account theage, general health, and nutritional status of the subject. Othercompositions' of the agent of the invention and further comprising,e.g., the macromolecular fraction of the defatted milk fat globulemembrane and the acidic fraction, are also contemplated herein.

Another aspect of this invention comprises a method of retarding theonset of, or countering, infantile gasteroenteritis associated withrotavirus infection comprising administering to an infant or child inneed of the treatment a composition comprising an anti-rotavirusinfection effective amount of an agent selected from the groupconsisting of defatted human milk fat globules, skim milk, the humanmilk macromolecular fraction, curd, whey, the human milk mucin-70 Kdapp.MW glycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kd app.MWglycoprotein, a polypeptide having the rotavirus-binding specificity ofthe 46 Kd app.MW HMFG glycoprotein, and mixtures thereof, and optionallya pharmaceutically-acceptable carrier and/or other agents and infantfoodstuffs such as formula, milk, and the like, as described above. Theabove method may be used for the prophylaxis of the disease,particularly where demographic and public health information suggestssignificant risk of infection. When symptoms indicate the onset ofinfection, the method may also be applied therapeutically.

The agent of this invention may be present in the infant formula in anamount from 0.01 to 99.9 wt %, and more preferably about 0.1 to 2.0 wt %of the composition. Other amounts of the agent, however, may also beused. As this product is formulated for the prophylatic or therapeutictreatment of infants and children afflicted with or at risk of diarrhealconditions associated with rotaviral infection, the infant food productmay include varying amounts of infant formula, juices, foods, milk ormilk supplements, among others. This anti-diarrheic infant product mayalso include vitamin supplements, water, mineral and other nutritionaladditives, salts, buffers, flavoring compounds, diluents, thickeners,emulsifiers, preservatives, encapsulation agents, glycosidaseinhibitors, protease inhibitors, and anti-oxidants, such as would befamiliar to a person skilled in the art. The infant formula may also bemodified to include varying amounts of water and other-solutes to meetother clinical needs of the infant or child. The human milk componentsof this invention being routinely consumed and consisting of biogicalmolecules, their administration will neither require clinicalprecautions nor medically trained personnel. Accordingly, the presentproducts may be sold over the counter.

Also part of this invention is a method of retarding the onset of, orcountering, diarrhea associated with rotavirus infection in a subject'scells comprising administering to a subject in need of such treatment acomposition comprising an anti-rotavirus effective amount of an agentselected from the group consisting of defatted human milk fat globules,skim milk, the human milk macromolecular fraction, curd, whey, the humanmilk mucin-70 Kd app.MW glycoprotein-46 Kd app.MW glycoprotein complex,the 46 Kd app.MW glycoprotein, a polypeptide having therotavirus-binding specificity of the 46 Kd app.MW HMFG glycoprotein andmixtures thereof, and optionally a pharmaceutically-acceptable carrierand/or a foodstuff. Because of minimal side effects associated with theagent used in this method, the agent may also be administered fordiarrheal symptoms regardless of etiology to prevent secondary outbreaksassociated with rotavirus infection.

This invention also provides a method of retarding the onset of, orcountering, diarrhea associated with rotavirus infection in animmunodeficient subject comprising administering to an immunodeficientsubject an anti-rotavirus effective amount of an agent selected from thegroup consisting of defatted human milk fat globules, the human milkmacromolecular fraction, skim milk, curd, whey, the human milk mucin-70Kd app.MW glycoprotein-46 Kd app.MW glycoprotein complex, the 46 Kdapp.MW glycoprotein, a polypeptide having the antibody-bindingspecificity of the about 46 Kd app.MW HMFG glycoprotein, and mixturesthereof, optionally comprising a pharmaceutically acceptable carrierand/or foodstuffs as described above. Such immunodeficiencies may resultfrom genetic dysfunction, organ transplant, disease induced conditionsor as a consequence of medical treatment with drugs, among others.

Other agents that may be added to the composition for this particularapplication are bulking agents, carbon black, high fiber additives,encapsulation agents, protease inhibitors, glycosidase inhibitors, andcarrier lipids, optionally micellar, among others. These may be presentin amounts known in the art.

Specific applications of the above method are in cases of, e.g.,transplants such as bone marrow, kidney, heart and other organtransplants. Transplant patients receiving immunosuppressant drugs mayalso benefit from this anti-diarrheic treatment.

The above preventative and therapeutic methods may be practiced byadministering the agent provided herein as part of an anti-diarrheiccomposition also comprising a carrier or a product such as a foodstuff,as described above. Suitable foodstuffs are milk, juices, cereals,powdered grains, candies, confections, cookies, meats, vegetables andfruits, put through a blender or otherwise processed, and crackers,among others.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein for purposes of illustration only and are not intended to belimiting of the invention or any embodiment thereof, unless sospecified.

EXAMPLES

Example 1: Source of Human Milk

Human milk was obtained from 30 healthy, lactating women donors to theCentral Massachusetts Regional Milk Bank, Worcester, Mass. The donorswere chosen to represent a wide range of maternal ages ranging from 20to 37 years (average: 28±4 years), parity: 9 primiparous, 13secundiparous, 5 tertiparous, 3 quadriparous with a 0 to 14 monthslactation period (average:6.0±3.8 months postpartum). The milk wasgenerally expressed in the morning by means of a mechanical pump, pooledand used to isolate the human milk components utilized below.

Example 2: Isolation of Oligosaccharide Components from Human Milk

The oligosaccharide fraction of human milk was prepared using the methodof Newburg et al. (Newburg, D. S., Pickering, L. K., McCluer, R. H., andCleary. T. G., "Fucosylated Oligosaccharides of Human Milk ProtectSuckling Mice from-Heat-stable Enterotoxin of Escherichia coil"J.Infect. Dis. 162:1075-1080 (1990)).

Briefly, 1.5 l of human milk were centrifuged at 4° C. at 3,000×g for 1hr to separate the cream from the skim milk. The cream was removed, andthe skimmed milk was filtered through glass wool. The filtrate was thenmixed with an equal volume of ice-cold acetone, stirred overnight at 4°C. and centrifuged at 3,000×g for 45 min to obtain a clear supernatant.The clear supernatant was concentrated by rotary evaporation at 40° C.to a volume of 300 ml and applied to a 1-L charcoal-Celite column (50%charcoal, 50% celite, Fischer Scientific, Boston, Mass.).

The column was extensively washed with distilled water and 5% aq.ethanol, and the oligosaccharide fraction was then eluted from thecolumn with 50% aqueous ethanol. The eluate was passed through an AG1-X2 anion-exchange resin (BioRad, Richmond, Calif.) to yield a neutraloligosaccharide fraction. The acidic oligosaccharides were eluted fromthe resin with 1M formic acid, and then dialyzed and lyophilized. Allfractions were stored at -70° C. until used.

Example 3: Isolation of Lipid components from Human Milk

Whole human milk was extracted with 20 volumes of chloroform/methanol(2:1 ), filtered, and the filtrate evaporated. The lipids extracted inthe chloroform phase were-then partitioned between hexane and 88%aqueous ethanol phases, free fatty acids being extracted in the hexanephase. Free fatty acids were removed from the hexane fraction by washingwith methanolic ammonia, and recovered by acidifying and extracting theprotonated fatty acids with hexane. The ethanol fraction was taken todryness, and subjected to Folch partition with chloroform/methanol/water(8:4:3:v:v:v:) to obtain upper phase and lower phase lipid fractions.

The upper phase lipids were isolated on a sepralyte C 18 (40 nm)reverse-phase column (American Bioanalytical, Natick, Mass.), and thenseparated on DEAE Sepharose (Pharmacia, Uppsala, Sweden) into neutralglycolipids and anionic glycolipids referred to as the "neutral" and"ganglioside" fractions. The Folch lower phase lipids were separated ona Unisil silicic acid column (Clarkson Chemical, Williamsport, Pa. toisolate the lower phase glycolipid fraction.

Example 4: Isolation of Macromolecular Fraction From Human Milk

Whole human milk was frozen and thawed three times and subsequentlysonicated for 20 minutes to disrupt the milk fat globules. Thecream-derived lipids, mainly triglycerides, were separated from the milkby centrifugation at 3,000×g for 1 hour at 4° C. and removed byfiltration through glass wool. The resulting skim milk was passedthrough a 300,000 Dalton molecular weight cut-off ultrafilter membranes.The retentate was washed three times with distilled water bydiafiltration to remove small proteins. This retentate was then dialyzedto remove any residual molecules smaller than 40 Kd app.MW, and thenlyophilized.

The specific activity was calculated as the amount of protein from wholepooled human milk representing the minimum inhibitory concentration(MIC₅₀) divided by the amount of protein from a purified fraction thatrepresented the MIC₅₀.

The specific activity (Sa) of a given purified fraction was calculatedfrom the following algebraic formula. ##EQU1## wherein the specificactivity is obtained by dividing the concentration of protein from theoriginal unprocessed whole pooled milk protein fraction that produces50% inhibition [WMP_(MIC50) ] by the concentration of protein from thepurified fraction necessary to produce 50% inhibition in the same assay[x_(MIC) 50 ].

Example 5: Separation of Human Milk Components by Isoelectric Focussing

Isoelectric focussing was performed in a Rotofor apparatus (BioRad,Richmond, Calif.). The macromolecular fraction, prepared as describedabove, was made 1% with ampholytes, pH 3-10, in 50 ml distilled water,and was resolved into 20 fractions at 14 W for 4 hrs. The pH of eachfraction was measured with a standard pH electrode, and each fractionwas then dialyzed against distilled water, and lyophilized. Eachfraction was then reconstituted with sterile phospate-buffered saline(PBS) such that each purified material was at the concentration at whichit was found in the original pooled milk sample. The active fractionswere pooled and refocussed as above, but over a pH gradient of 3.9 to7.1 over 20 fractions.

The presence of glycoproteins immunologically related to human milkmucin: the 46 Kd app.MW glycoprotein, and the 70 Kd app.MW glycoprotein,in the highly focussed fractions was then determined as described byPeterson et al. (Peterson, J. A., et al., Hybridoma 9:221-235. (1990)).The presence of the glycoproteins was determined by depositing analiquot of each fraction onto a well of a microtiter plate and adding tothe plates, Mc5, a monoclonal antibody which specifically binds milkmucin, Mc13, a monoclonal antibody that specifically binds to the 70 Kdapp.MW glycoprotein and, Mc16, a monoclonal antibody that bindsspecifically to the 46 Kd app. Mw glycoprotein. Where present, theglycoproteins bound to the wall and their binding to the antibody wasquantitated by a subsequent reaction with peroxidase labeled anti-mouseIgG, and o-phenylene diamine H₂ O₂ substrate. The mucin-relatedglycoprotein activity was expressed as the optical density at 450 nmgenerated by reaction in the wells coated with the milk fractions minusthe optical density generated by reaction in a control well without milkcomponents. The fractions binding the antibody were pooled anddesignated as acidic glycoprotein.

The presence of IgG and secretory IgA immunoglobulins in the fractionsobtained by isoelectric focussing was tested by the solid phase enzymeimmunoassay procedure of Yolken et al modified as described below.(Yolken, R. H., et al., J. Pediatr. 93:916-921 (1978)).

Briefly, the Yolken et al assay comprises binding immuno-globulins tothe surface of a solid support, reacting the immunoglobulins withenzyme- or otherwise-labeled anti-immunoglobulin antibodies, andreacting, e.g., the bound enzyme with a color-changing substrate, sothat a change in the wavelength in the color-producing region permitsthe quantitation of the specific immunoglobulin present in the initialsample.

The presence of trypsin inhibitory activity in the fractions was alsotested using a benzoyl-DL-arginine-p-nitroanilide (BAPNA) substrate inaccordance with Vonderfecht et al. (Vonderfecht, S. L., et al, J. Clin.Invest. 82:2011-2016 (1988)).

Example 6: Isolation of Human Milk Mucin Complex

The human milk mucin complex present in delipidated or defatted humanmilk fat globules (Peterson, J. A. et al., Hybridoma 9:221-235. (1990))was isolated by affinity chromatography using the Mc5 monoclonalantibody that recognizes the tandem repeat region of the human milkmucin. (Peterson, J. A., et al., in Breast Epithelial Antigens:Molecular Biology to Clinical Applications. R. L. Ceriani, ed., PlenumPublications, New York, pp. 55-68 (1991)). The Mc5 monoclonal antibodywas conjugated with cyanogen bromide activated Sepharose-4B beads(Pharmacia, Uppsala, Sweden) at a ratio of 1 mg of IgG/ml swollen beads.The antibody coated beads were washed and suspended in PBS containing0.3%. Triton X-100, 10% bovine serum and 0.1% sodium azide. Delipidatedhuman milk fat globule was dissolved in the same buffer, sonicated, andincubated with antibody coated beads overnight at 4° C. The beads werealternately washed 3 times with 0.1M Na acetate,. 1M NaCl, pH 4.0, and0.1M Tris buffer, pH 8.0, and twice with PBS containing 0.3% TritonX-100. The bound mucin complex was eluted with 3M sodium isocyanate andconcentrated by dialysis.

Example 7: Separation of Human Milk Mucin Components

The milk mucin, and the 46 Kd app.MW and 70 Kd app.MW glycoproteincomponents of the milk mucin complex were separated by fractionation ofdefatted human milk fat globule under reducing conditions (0.5%β-mercaptoethanol), followed by electrophoresis in a 3%-15% SDSpolyacrylamide gel. One lane was stained with Coomassie blue to identifythe components while the remainder of the gel was frozen. Bandscontaining the reduced human milk mucin of approximately 400 Kd app.MW,the 70 Kd app.MW glycoprotein component, and the 46 Kd app.MWglycoprotein component were excised from the gel, separatelyhomogenized, placed in dialysis bags and eluted from the gel bands byelectrophoresis in the presence of 0.5% β-mercaptoethanol and 2Mdithiothreitol.

The eluted proteins were separated from the gel by centrifugation. Eachprotein was characterized by its binding to specific monoclonalantibodies (Mc5 for milk mucin, and Mc13 for the 70 Kd app.MWglycoprotein and and Mc16 for the 46 Kd app.MW glycoprotein) (Petersonet al (1990), supra).

Example 8: Desialylation of Human Mucin Components

Sialic acid was removed in one set of samples from the componentspurified in Example 7 by chemical hydrolysis using a previouslydescribed method (Gibbons, R. A., Biochem. J. 89:380-391 (1963); Gyorky,G.et al., Can. J. Biochem. 43:1807-1811 (1965); Jourdian, G. W., et al.,J. Biol Chem. 246:430-435 (1971)).

Briefly, samples containing 1 to 10 mg of human milk glycoproteinspurified as described above, were suspended in 1 ml 0.08N H₂ SO₄, anddigested for 1 hour at 80° C. After rapid cooling on ice, the sampleswere neutralized with 100 μl of 0.8M NaOH, exhaustively dialyzed andlyophilized.

The glycosidic linkages of the sialic acid residues of glycoproteins areuniquely sensitive to mild acid hydrolysis. The specificity of achemical hydrolysis of glycosidic linkages is dependent upon thereaction conditions which are employed. Since 0.1N hydrochloric acidresults in the hydrolysis of 20% of acylneuraminic acids, dilutesulfuric acid hydrolysis of sialic acid is preferred. The release ofsialic acids from human serum and brain was optimal with 0.1N sulfuricacid at 80° C. for 1 hour, depending on the concentration of sialic acidin the sample (Suennerholm, L., Acta Chem. Scand. 12:547-584 (1958)).Thus, the conditions for acid hydrolysis (0.08N H₂ SO₄, 80° C., 60minutes) used effect complete release of sialic acid, a partial releaseof approximately 3% of the fucose and negligible release of othersugars. (Gyorky, G. et al., Can J. Biochem. 43:1807-1811 (1965).).

Example 9: Effect of Desialylation on Binding of the 46Kd app.MW HMFGGlycoprotein to Rotavirus

The binding inhibition assay was conducted as shown in Example 12. Theseparation of sialic acid by hydrolysis from the 46 Kd app.MWglycoprotein reduced its ability for binding to various rotavirusstrains. Since the rotaviruses do not hemagglutinate erythrocytes orbind to other sialic acid-containing compounds, it is thus possible thatthe linkages involved in the viral binding to the apparent molecularweight 46 Kd app.MW glycoprotein are different from those present onerythrocytes and other sialic acid-containing compounds.

Another possibility is that the rotavirus does not bind directly to asialic acid-containing portion of the target molecule, and that theremoval of sialic acid results in a conformational alteration of the 46Kd app.MW binding epitope. It is unlikely that the chemical hydrolysisof the 46 Kd app.MW glycoprotein eliminates the reactivity of the MC16monoclonal antibody to the resulting glycoprotein since this antibodyrecognizes a peptide domain of the glyceprotein (Peterson, J. A., et al.Hybridoma 9:221-235 (1990)). Furthermore, the monoclonal antibody wasdemonstrated to still bind to the 46 Kd app.MW glycoprotein followingchemical desialylation.

Example 10: Isolation of Various Strains of Rotavirus

The rhesus rotavirus (RRV) strain MMU 18006, the simian rotavirus strainSA-11, the mouse rotavirus strain (EDIM) and the human rotavirus strainsWa, DS-1, P, and ST3 were propagated in MA-104 cells using the methoddescribed by Yolken, R. H., et al. (Yolken, R. H., et al., "Sialic AcidGlycoproteins Inhibit the in Vitro and In Vivo Replication ofRotaviruses", J. Clin. Invest. 79:148-154 (1987)).

The serotype of the neutralization protein VP7 was determined for allstrains. The simian strains were shown to belong to serotype 3, whereasthe human strains Wa, Ds-1, P and ST3 belong to serotypes 1, 2, 3, and4, respectively (Moshino, Y., et al., J. Infect, Dis. 149:694-702(1984)).

Example 11: Binding of Various Rotavirus Strains to Mucin

Briefly, the above viruses were diluted in PBS to concentrations ofapproximately 10³ pfu/ml and immobilized onto wells of microtiter platescoated with methylated bovine serum albumin as described by Ceriani etal. (Ceriani, R. L, in:Monoclonal Antibodies and Functional Cell Lines.Progress and Applications, Bechtol, K. B., McKern, T. J. and Kennett,R., eds., Plenum Press, New York, pp. 398-402 (1984)).

The control wells were coated with an equivalent concentration ofuninfected MA-104 cells under identical conditions. After washing thewells with PBS-Triton-X100, the viruses and the controls were reactedwith the mucin components at a concentration of 0.5 μg/well.

The binding of the mucin components to the virus and the uninfectedcontrol cells was determined by reaction with monoclonal antibodies Mc5,Mc13, and Mcl 6 that are specific for mucin, the 70 Kd app.MWglycoprotein, and the 46 Kd app.MW glycoprotein components of the nativemucin complex, respectively. After washing with. PBS-Triton X-100, ¹²⁵ Ilabelled anti-murine IgG was added. (Peterson et al (1990), supra). Thebinding was quantitated by measuring the ¹²² I- labeled anti-murine IgGattached to well-bound monoclonal antibodies.

Each sample was measured in quadruplicate and a binding ratio wascalculated for each component by dividing the average number of countsgenerated in the virus-coated well by the average number of countsgenerated in the control wells with uninfected cells.

Example 12: Inhibition by Human Milk Mucin Fractions of RotavirusInfection

The human milk fractions prepared as described above were diluted tovarying concentrations in Eagle's Modified Essential Medium (EMEM)containing 0.5 to 1 μg/ml porcine trypsin and approximately 100 pfu ofthe MMU rotavirus strain. The cells were allowed to adsorb thevirus-milk factor mixture for 1 hr at 37° C. The cell monolayers werethen washed and covered with an overlay of agarose containing 0.5 μg/mltrypsin plus the anti-viral milk factor at the same concentration usedfor the adsorption step. The samples were incubated for approximately 5days at 37° C., a second agarose overlay containing neutral red dye wasadded, and the stained plaques counted.

The percentage inhibition was calculated for each concentration of allmilk components used as follows.

Percentage inhibition=100x(1-(P_(f) /P_(c))),

wherein

P_(f) is the number of plaques generated in cells infected with virusincubated with the milk factor, and

P_(c) is the number of plaques generated in the virus infected cells inthe absence of added milk fractions.

Each fraction was initially tested at a concentration equivalent to thatin which it is found in the human milk pool. The samples whichdemonstrated inhibition were then retested at 10-fold dilutions. Theminimum inhibitory concentration (MIC₅₀) for each fraction wascalculated by interpolating the minimum concentration required for the50% inhibition of plaque generation.

Example 13: Inhibition by Milk Mucin of In Vivo Rotavirus Infection

A murine rotavirus (EDIM) strain responsible for epizootic diarrhea ininfant mice was purified by ultracentrifugation through CsCl. Theability of the macromolecular fraction and the acidic glycoproteinfraction prepared as described in Examples 4 and 5 above, respectively,to prevent diarrhea associated with EDIM in suckling mice was tested.The EDIM rotavirus was separately incubated at a concentration of 10ID₁₀₀ /ml with the different milk fractions and with controlpreparations at a concentration of 100 μg/ml.

The control preparations contained bovine milk-based infants' formula(Similac, Ross Laboratories), or minimum essential media.

Following incubation for 30 minutes at 37° C., 100 μl of the milk-virusmixture were fed to individual mice in separate litters of sucklingmice. To minimize variations among litters, the suckling mice werepooled and then randomly divided among the dams immediately prior toinoculation. The suckling mice remained with these dams throughout thecourse of the study. The development of diarrhea was observed for 3-5days following infection with the virus. The suckling mice continued toreceive milk from their mothers during the course of the study.

The macromolecular fraction of human milk totally prevented thedevelopment of rotavirus gastroenteritis in the animal model.Furthermore, feeding the milk acidic glycoprotein component along withrotavirus resulted in>90% protection against symptomaticgastroenteritis. On the other hand, neither infant formula based oncow's milk or tissue culture media afforded any measurable protectionagainst symptomatic infection.

Example 14: Inhibition of Rotavirus Infection in Suckling Mice by MilkComponents

0.1 ml aliquots of the EDIM murine rotavirus containing x10⁶ pfu/ml wereseparately incubated with individual milk components or controlsolutions and fed to suckling mice. The macromolecular and acidicglycoprotein human milk components were tested at concentrations of100μg/ml and the bovine milk formula and the tissue culture media weretested without dilution. The following exposure to the rotavirusdevelopment of diarrhea in the animals was observed for 3-5 days.

The macromolecular fraction of human milk prevented the development ofrotavirus-induced diarrhea by nearly 100% in suckling mice. When fed tothe suckling mide infected with rotavirus the acidic glycoproteincomponent of human milk resulted in greater than 90% protection againstdiarrheal symptoms.

Neither an infant formula based on cow's milk nor tissue culture mediaby themselves provided a significant degree of protection againstsymptomatic rotavirus infection.

Example 15: Determination of Milk Components Having Anti-RotavirusInfection Activity

The oligosaccharide, lipid, and macromolecular protein components ofhuman milk were fractionated and reconstituted to a concentrationten-fold greater than in the original milk as described by Newburg, D.S., et al. (Newburg, D. S., et al., Pediatric Res. 31:22-28 (1992)). Theability of each of the fractions to inhibit the replication of the SA-11rotavirus strains in tissue culture was then tested at ten-fold theirconcentration found in human milk, and at logarithmic dilutions, e.g.,1:10, 1:100, 1:1,000, 1:10,000, etc.

The lipids extracted from whole human milk were fractionated intoganglioside, polar neutral glycolipid, non-polar glycolipid,triglyceride and fatty acid fractions as described by Newburg, D. S., etal, (Newburg, D. S., et al. (1992), supra)). None of the lipidcomponents of the milk displayed significant anti-rotavirus activity atany concentration.

In addition, the milk oligosaccharides were separated into neutral andacidic oligosaccharide fractions, both of which aisc failed to showanti-rotaviral activity.

The macromolecular fraction (>300 Kd app.MW MW) was shown to inhibit thereplication of the prototype SA-11 rotavirus strain (VP7 serotype 3)with a minimum inhibitory concentration of approximately 100 μg/ml. Thisis a 3-fold increase in activity when compared to the original wholehuman milk pool.

The macromolecular fraction of human milk was further fractionated byhigh-resolution preparative isoelectric focussing as described byNewburg et al. (Newburg, D. S., et al., Pediatr. Res. 31:22-28 (1992)).The inhibitory activity of the rotavirus was confined to fractions witha pi of 4.0 to 4.6. These fractions did not contain immunoglobulinsdetectable by solid phase immunoassay or protease inhibitory activitymeasured by reaction with trypsin and benzoyI-DL-arginine-p-nitroanilide(BAPNA) substrate.

These acidic fractions with anti-rotaviral activity also reacted withmonoclonal antibodies directed at the human milk mucin complex. Theacidic fractions with anti-rotaviral activity were pooled and designatedas the acidic-glycoprotein fraction of human milk. This acidicglycoprotein fraction inhibited the replication of the SA-11 rotavirusat a concentration of 8 μg/ml (1:500 dilution from its originalconcentration in milk). This represents a 38-fold increase in specificactivity.

Example 16: Inhibition of Rotavirus Infection by the Purified MucinComplex

The active human milk mucin complex of the invention was affinitypurified from defatted human milk fat globule utilizing the monoclonalantibody Mc5 as described above in Example 6. The affinity-purified milkmucin complex inhibited the infection of the simian SA-11 virus inMA-104 cells at a concentration of 0.1 μg/ml (approximately 2×10⁻¹⁰mol/l, based on a molecular weight of 500,000). This is a 3,000-foldincrease in specific activity over whole milk. Thus, the affinitypurified complex shows extremely potent activity to block rotavirusinfection. Furthermore, the complex binds directly to rotavirus, asmeasured using the solid phase binding assay of Example 11.

Example 17: Inhibition of Human Rotavirus Infection by Purified MucinComplex - Effect of Sialic Acid

The mucin complex was deglycosylated by treatment with hydrogen fluoridefor 3 hours at room temperature as described by Mort and Lainport (Mortand Lamport. Anal Biochem. 82:238-309 (1977)). These conditions resultin the hydrolysis of most glycosidic bonds as well as a substantialreduction in the inhibition of rotavirus activity by the complex. Nosignificant rotavirus inhibition was measurable at 10 μg/mldeglycosylated mucin, the highest concentration tested.

Various human rotavirus strains were incubated with the human milkmacromolecular glycoprotein fraction at a concentration of 250 μl/ml andpropagated in MA-104 cells as described herein. The macromolecularglycoprotein fraction of human milk, at a concentration of 250 μl/ml,inhibited approximately 55-80% the in vitro replication of 4 humanstrains of rotavirus (WA, OS-1, P, St-3).

The viruses were also incubated with the desialylated macromolecularfraction of the milk mucin complex, followed by propagation in MA-104cells. For all 4 human virus strains, the inhibition of rotavirusinfection was reduced (WA, DS-1) or abolished (Pt, St-3) by the chemicaldesialylation of the milk glycoprotein.

Example 18: Specific Binding of Rotavirus to the 46 Kd app.MW HMFGGlycoprotein

The native, affinity purified milk mucin complex was found to bindspecifically to cells infected with the simian SA-11 rotavirus.

The milk mucin complex was fractionated under reducing conditions, whichresulted in the dissociation of the complex in its components. Followingfractionation, the isolated milk mucin components were exposed torotavirus and to uninfected cells (control) bound to microtiter plates.The reduced 46 Kd app.MW glycoprotein of the milk mucin complexseparated by gel electrophoresis retained substantial viral bindingactivity. The rotavirus specifically bound the 46 Kd app.MW glycoproteincomponent of the mucin complex whereas no significant binding was foundto occur with the 70 Kd app.MW glycoprotein component or with thereduced residual macromolecular mucin in the absence of the 46 Kd app.MWor the 70 Kd app.MW glycoprotein components.

The 46 Kd app.MW component bound to the simian rotavirus strain RRV (VP7serotype 3) and to human rotavirus strains Wa, DS-1, P, and ST3 (VP7serotypes 1, 2, 3, and 4, respectively). The 46 Kd app.MW HMFGglycoprotein bound to each of the human strains with approximately equalavidity. The binding of the 46 Kd app.MW HMFG glycoprotein to the humanvital strains did not differ significantly from its binding to thesimian strain.

The hydrolysis of sialic acid in the 46 Kd app.MW glycoprotein resultedin a substantial decrease in the binding of the 46 Kd app.MWglycoprotein to the rhesus rotavirus and the other human rotavirusstrains (all p<0.01; 2 tailed t-test).

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as novel in Letters Patent of the United States is: 1.An anti-viral composition comprisingan agent selected from the groupconsisting of defatted human milk fat globule, the haman milkmacromolecular fraction, the human milk mucin-70 Kd apparent MW HMFGglycoprotein:46 Kd apparent MW HMFG glycoprotein complex, the 46 Kdapparent MW HMFG glycoprotein, a polypeptide comprising an amino acidsequence having the rotavirus-binding specificity of the 46 Kd apparentMW HMFG glycoprotein, deglycosylated proteins thereof, mixtures thereof,and mixtures thereof with human skim milk, curd, or whey; and atherapeutic agent, or mixtures thereof.
 2. The composition of claim 1,further comprising an additive selected from the group consisting ofcoatings, sustained release additives, carrier lipids, lubricants,bulking agents, carbon black, high fiber additives, encapsulationagents, nutritional additives, buffers, salts, flavoring agents,diluents, thickeners, emulsifiers, preservatives, antioxidants, binders,excipients, anti-clumping agents, sweeteners, and mixtures thereof. 3.The composition of claim 1, further comprising a pharmaceuticallyacceptable carrier.
 4. The composition of claim 1, further comprising afoodstuff, wherein the agent is dispersed.
 5. The composition of claim4, wherein the foodstuff comprises milk, whey, curd, infant formula,juices, cereals, candy, chewing gum, cookies, crackers, vegetables,meats, fruits, or mixtures thereof.
 6. The composition of claim 1,wherein the agent comprises a polypeptide comprising an amino acidsequence having the rotavirus-binding specificity of the 46 Kd apparentMW HMFG glycoprotein, deglycosylated proteins thereof, or mixturesthereof.
 7. The product of claim 6, wherein the agent comprises the 46Kd apparent MW HMFG glycoprotein, deglycosylated proteins thereof, ormixtures thereof.
 8. The composition of claim 1, wherein the agent ispresent in amount of 0.01 to 99.9 wt %.
 9. An anti-vital kit comprising,in separate sterile containers,the agent of the composition of claim 1;a therapeutic agent, and-mixtures thereof; and instructions for use ofthe kit.
 10. A method of retarding or treating rotavirus infection in asubject, comprising administering to the subject a compositioncomprising an anti-rotavirus effective amount of an agent selected fromthe group consisting of defatted human milk fat globule, the human milkmacromolecular fraction, the human milk mucin-70 Kd apparent MWglycoprotein-46 Kd apparent MW glycoprotein complex, the 46 Kd apparentMW glycoprotein, a polypeptide comprising an amino acid sequence havingthe rotavirus-binding specificity of the 46 Kd apparent MW HMFGglycoprotein, deglycosylated proteins thereof, mixtures thereof, andmixtures thereof with human skim milk, curd, or whey.
 11. The method ofclaim 10, wherein the agent comprises a polypeptide comprising an aminoacid sequence having the rotavirus-binding specificity of the 46 Kdapparent MW HMFG glycoprotein, deglycosylated proteins thereof, ormixtures thereof.
 12. The method of claim 11, wherein the agentcomprises the 46 Kd apparent MW HMFG glycoprotein, deglycosylatedproteins thereof, or mixtures thereof.
 13. The method of claim 12,wherein the agent comprises synthetic 46 Kd apparent MW HMFGglycoprotein, deglycosylated proteins thereof, or mixtures thereof. 14.The method of claim 10, wherein the agent is administered to the subjectin an amount of 0.1 to 1000 mg/kg body weight/day.
 15. The method ofclaim 10, wherein the composition further comprises a carrier.
 16. Themethod of claim 15, wherein the carrier comprises a pharmaceuticallyacceptable carrier.
 17. The method of claim 10, wherein the carriercomprises a foodstuff, in which the agent is dispersed.
 18. The methodof claim 17, wherein the foodstuff comprises milk, whey, curd, infantformula, juices, cereals, candy, chewing gum, cookies, crackers,vegetables, meats, fruits, or mixtures thereof.
 19. The method of claim10, wherein the agent is present in the composition in an amount of 0.01to 99.9 wt %.
 20. The method of claim 10, wherein the subject is anormal subject.
 21. The method of claim 10, wherein the subject is atransplant subject.
 22. The method of claim 21, wherein the transplantsubject is a bone marrow transplant subject.
 23. The method of claim 10,wherein the subject is an immunodeficient subject.
 24. The method ofclaim 23, wherein the immunodeficient subject is afflicted with agenetic immune deficiency.
 25. The method of claim 23, wherein theimmunodefficient subject is afflicted with an acquired immunedeficiency.
 26. The method of claim 23, wherein the immunodefficientsubject's immune system is depressed or suppressed by dragadministration.
 27. The method of claim 10, wherein the subject is anelderly subject.
 28. The method of claim 10, wherein the subject is aninfant or a child.
 29. The method of claim 10, further comprisingadministering to the subject an anti-viral agent.
 30. The method ofclaim 29, wherein the anti-viral agent is selected from the groupconsisting of α-interferon, ribonucleases, oligosaccharides,anti-rotaviral immunoglobulins, trypsin .inhibitors, anti-rotaviralserum, anti-rotavirus vaccines, and combinations thereof.
 31. The methodof claim 10, wherein the composition further comprises an additiveselected from the group consisting of therapeutic agents, coatings,sustained release additives, carrier lipids, bulking agents, carbonblack, high fiber additives, encapsulation agents, nutritionaladditives, buffers, salts, flavoring agents, diluents, thickeners,emulsifiers, preservatives, anti-oxidants, binders, excipients,anti-clumping agents, lubricants, sweeteners, and mixtures thereof. 32.The method of claim 10, wherein the subject is an animal.
 33. The methodof claim 32, wherein the subject is a human.
 34. A method of retardingor treating diarrhea associated with rotavirus infection in a subjectcomprising administering to the subject a composition comprisinganti-diarrhea effective amount of an agent selected from the groupconsisting of defatted human milk fat globule, the human milkmacromolecular fraction, the human milk mucin-70 Kd apparent MW HMFGglycoprotein-46 Kd apparent MW HMFG glycoprotein complex, the 46 Kdapparent MW HMFG glycoprotein, a polypeptide comprising an amino acidsequence having the rotavirus-binding specificity of the 46 Kd apparentMW HMFG glycoprotein, deglycosylated proteins thereof, mixtures thereof,and mixtures thereof with human skim milk, curd, or whey.
 35. A methodof retarding or treating infantile gastroenteritis associated withrotavirus infection comprising administering to an infant in need of thetreatment a composition comprising an anti-infantile gastroenteritiseffective amount of an agent selected from the group consisting ofdefatted human milk fat globule, the human milk macromolecular fraction,the human milk mucin-70 Kd apparent MW glycoprotein-46 Kd apparent MWglycoprotein complex, the 46 Kd apparent MW glycoprotein, a polypeptidecomprising an amino acid sequence having the rotavirus-bindingspecificity of the 46 Kd apparent MW HMFG glycoprotein, deglycosylatedproducts thereof, mixtures thereof, and mixtures thereof with human skimmilk, curd, or whey.
 36. A method of retarding or treating diarrheaassociated with rotavirus infection comprising administering to asubject in need of the treatment a composition comprising ananti-diarrhea effective amount of an agent selected from the groupconsisting of defatted human milk fat globule, the human milkmacromolecular fraction, the human milk mucin-70 Kd apparent MWglycoprotein-46 Kd apparent MW glycoprotein complex, the 46 Kd apparentMW glycoprotein, a polypeptide comprising in amino acid sequence havingthe rotavirus-binding specificity of the 46 Kd apparent MW HMFGglycoprotein, deglycosylated products thereof, mixtures thereof, andmixtures thereof with human skim milk, curd, or whey.
 37. A method ofretarding or treating diarrhea associated with rotavirus infection in animmunodeficient subject comprising administering to an immunodeficientsubject in need of the treatment a composition comprising ananti-diarrhea effective amount of an agent selected from the groupconsisting of defatted human milk fat globule, the human milkmacromolecular fraction, the human milk mucin-70 Kd apparent MWglycoprotein-46 Kd apparent MW glycoprotein complex, the 46 Kd apparentMW glycoprotein, a polypeptide comprising an amino acid sequence havingthe rotavirus-binding specificity of the 46 Kd apparent MW HMFGglycoprotein, deglycosylated products thereof, mixtures thereof, andmixtures thereof with human skim milk, curd, or whey.
 38. The method ofclaim 25, wherein the subject afflicted with an acquired immunedeficiency is an HIV+ subject.
 39. The method of claim 23, wherein theimmunodefficient subject is afflicted by a disease-inducedimmunodefficiency.
 40. The method of claim 21, wherein the transplantpatient is further administered immunosuppressant therapy.
 41. Themethod of claim 10, further comprising administering to the subject anagent selected from the group consisting of immunosuppressant drugs,α-interferon, ribonucleases, vitamins, minerals, milk lipids, proteaseinhibitors, glycosidase inhibitors, and mixtures thereof.
 42. A methodof retarding or treating rotavirus infection in a subject, comprisingadministering to the subject the composition of claim 1, in an amounteffective to deliver an anti-rotavirus effective amount of the agent.43. A method of retarding or treating diarrhea associated with rotavirusinfection in a subject comprising administering to the subject thecomposition of claim 1, in an amount effective to deliver ananti-diarrhea effective amount of the agent.
 44. A method of retardingor treating infantile gastroenteritis associated with rotavirusinfection comprising administering to an infant in need of the treatmentthe composition of claim 1, in an amount effective to deliver ananti-infantile gastro enteritis effective amount of the agent.
 45. Amethod of retarding or treating diarrhea associated with rotavirusinfection comprising administering to a subject in need of the treatmentthe composition of claim 1, in an amount effective to deliver ananti-diarrhea effective amount of the agent.
 46. A method of retardingor treating diarrhea associated with rotavirus infection in animmunodeficient subject comprising administering to an immunodeficientsubject in need of the treatment the composition of claim 1, in anamount effective to deliver an anti-diarrhea effective amount of theagent.
 47. The composition of claim 1, wherein the therapeutic agent isselected from the group consisting vitamins, minerals, immunosuppressantdrugs, α-interferon, ribonucleases, milk lipids, protease inhibitors,glycosidase inhibitors, lubricants, and mixtures thereof.
 48. Thecomposition of claim 1, in dehydrated form.
 49. The composition of claim1, wherein the active ingredient is provided in coated form.
 50. Thecomposition of claim 1, provided in coated form.
 51. The composition ofclaim 1, for use in the form of a tablet, troche, capsule, elixir,suspension, syrup, or wafer.
 52. The composition of claim 1, in the formof a tablet, troche, capsule, elixir, suspension, syrup, or wafer. 53.The composition of claim 1, in the form of a sustained releaseformulation.
 54. The composition of claim 1, in self-administrable form.55. The composition of claim 1, in unit form.
 56. The composition ofclaim 1, in bulk form.
 57. A food supplement, comprising the compositionof claim 1, wherein the additive comprises vitamins and minerals.
 58. Ananti-microbial product, comprising the composition of claim 1, whereinthe additive comprises at least one therapeutic agent.
 59. A product,comprising a baby or infant foodstuff; and the composition of claim 1.60. The product of claim 59, wherein the foodstuff comprises formula.61. The product of claim 60, in rehydratable form.
 62. The kit of claim9, further comprising an additive selected from the group consisting ofcoatings, sustained release additives, carrier lipids, bulking agents,carbon black, high fiber additives, encapsulation agents, nutritionaladditives, buffers, salts, flavoring agents, diluents, thickeners,emulsifiers, preservatives, anti-oxidants, binders, excipients,anti-clumping agents, lubricants, sweeteners, mixtures thereof, andcarriers.
 63. The kit of claim 9, wherein the therapeutic agent isselected from the group consisting of vitamins and minerals.